(1) Field of the Invention
The invention relates to a microscopy method and/or a microscope for generating a high-resolution image of a luminescent sample.
(2) Description of Related Art Including Information Disclosed Under 37 CFR 1.97 and 1.98
The examination of samples using microscopy is a broad technical field for which there are numerous technical solutions. The most varied of the known microscopy methods have developed from the area of classic light microscopy.
A classic area of application of light microscopy for examining biological preparations is luminescence microscopy. In this field, certain dyes (so-called phosphors or florophores) are used to specifically label samples, e.g. cell parts. The sample is illuminated with excitation radiation, as previously mentioned, and the luminescence light created thereby is detected with suitable detectors. Typically, the light microscope is equipped with a dichroic beam splitter together with block filters that divide the fluorescence beam from the excitation beam and enable separate monitoring. This process enables the representation of individual, differently colored parts of the cell in the light microscope. Of course, multiple parts of a preparation may also be marked with different dyes that are absorbed specifically at different structures of the preparation at the same time. This process is known as multiple luminescence. Samples can also be measured that exhibit luminescence on their own, without adding a marker sub stance.
Luminescence in this case, as it is generally known, is a generic term that describes phosphorescence and fluorescence; thus it includes both processes.
In addition, laser scanning microscopy (abbreviated as LSM) is also known to be used to examine samples; it reproduces only that plane that is in the focal plane of the lens from a three-dimensional illuminated image using a confocal detection layout (known as confocal LSM) or nonlinear sample interaction (so-called multiphoton microscopy). An optical cross-section is obtained and the detection of multiple optical cross-sections in various levels of the sample enables the subsequent generation of a three-dimensional image of the sample, composed of various optical cross-sections, with the assistance of a suitable data processing device. Therefore, laser scanning microscopy is suitable for examining thick preparations.
Various approaches have been developed in recent times for resolutions beyond the diffraction limits set by the laws of physics. These microscopy methods are characterized in that they provide users with a higher lateral and/or axial optical resolution as compared to a classic microscope. In this description, such microscopy methods are characterized as high-resolution microscopy methods, because they achieve a resolution beyond the optical diffraction limit. Diffraction-limited microscopes, on the other hand, are characterized as classic microscopes. They utilize known optical wide-field microscopy or laser scanning microscopy.
A high-resolution microscopy method is addressed in U.S. Pat. No. 6,909,105 B1. In this case, nonlinear processes are utilized by means of structured illumination. The saturation of the fluorescence serves to represent nonlinearity. A structured illumination effects a displacement of the object space spectrum relative to the transfer function of the optical system. More specifically, the displacement of the spectrum means that object space frequencies V0 are transmitted at a spatial frequency V0-Vm, where Vm represents the frequency of the structured illumination. At a given spatial frequency that is maximally transferable by the system, this enables the transfer of spatial frequencies of the object that are greater by the amount of the displacement frequency Vm than the maximum frequency of the transfer function. This approach requires Fourier filtering as a reconstruction algorithm for creating images and the evaluation of multiple views for an image. U.S. Pat. No. 6,909,105 B1, which is likewise fully incorporated by reference herein with respect to the corresponding description of the resolution microscopy method, thus uses structured wide-field illumination of the sample, for example via an amplitude/phase grating.
Fluorescence in the sample is also detected using wide-field microscopy. The grating is then placed into three different rotational positions, e.g. 0°, 120°, and 240°, and the grating is then displaced into three or more different positions within each rotational position. Wide-field microscopy is used on the sample in each of the displacements of the three rotational positions (i.e. for a total of at least 9 illumination states). Furthermore, the grating has frequencies as close as possible to the limit frequency that the optical layout used is capable of transferring. The above-mentioned displacement then takes place with the use of Fourier filtering, whereby particularly the 0 and +/−1 diffraction layouts in the images are evaluated. This microscopy method is also known as the SIM method.
An increase in the resolution is obtained with this principle when the structuring (e.g. by means of a grating) is intensive to the extent that the fluorescence in the sample achieves saturation in the bright areas. The structured illumination of the sample no longer has a sinusoidal distribution on the sample but rather an even higher harmonic one beyond the optical limit frequency due to the saturation effects. This additional development of the SIM method is also known as saturated pattern excitation microscopy (SPEM).
Further development of the SIM method can also be achieved with a line-type illumination that is perpendicular to the direction of the grating. A linear illumination is then achieved in which the grating structure is reproduced along the line. The line of the illumination is thus, on its part, structured by the grating. The line-type illumination enables confocal slit detection and thus another increase in resolution. This method is also abbreviated as SLIM.
The publication by C. Müller and J. Enderlein, “Image scanning microscopy,” Physical Review Letters, 104, 198101 (2010), addresses the SIM principle, but utilizes scanning of the sample with confocal illumination and detection with subsequent Fourier filtration. This principle is known as ISM. With this method, there are not nine orientations of a structured illumination but instead each scan position, i.e. each grating state when an image is scanned, corresponds to an illumination state and the structured illumination is a spot illumination of the sample.
The publication from T. Dertinger, et al., “Fast, background-free, 3-D super-resolution optical fluctuation imaging (SOFI),” PNAS (2009), page 22287-22292 and “Achieving increased resolution and more pixels with Super-resolution Optical Fluctuation Imaging (SOFI),” Opt. Express, 30 Aug. 2010, 18(18): 18875-85, doi: 10.1364/IE.18.018875 and S. Geissbuehler et al., “Comparison between SOFI and STORM,” Biomed. Opt. Express 2, 408-420 (2011), discloses a further high-resolution method of luminescence microscopy. This method utilizes the flashing properties of a fluorophore. If the fluorophores of a sample are flashing statistically independently of one another, a reproduction of the sample can be achieved through filtration with a so-called cumulant function as well as a significant increase in the resolution beyond the given physical resolution limit. An example of such a cumulant function is the second-order autocorrelation function. To generate a high-resolution image in this case, a series of individual images is acquired and combined to create a single image, which then has the higher resolution, using the cumulant function. This method is abbreviated as the SOFI method that stands for “Super-Resolution Optical Fluctuation Imaging.”
Combining multiple high-resolution microscopy methods is further known in prior art. Thus, for example, US 2011/0284767 A1 describes combining various high-resolution microscopy methods with the goal of being able to use the particular method that is optimum for each individual sample area in terms of resolution and measuring speed.